Pharmacokinetic properties of drugs are essential determinants of the pharmaceutical action drugs perform within the human body. In most cases, drugs need to be sufficiently long lived within the organism, yielding concentrations in suitable body compartments which are high enough for the display of the desired pharmacological function (e.g., binding of a drug to its receptor).
In many cases, desired pharmacokinetic properties of small drugs are a result of a certain binding capacity to albumin, which is the most abundant protein in blood, at concentrations of approx. 600 μM. Pharmaceutical companies can empirically screen for suitable albumin binding characteristics of their drug of interest. However, this approach is of limited utility, as in most cases the albumin binding properties of the molecule are intimately connected to the portion of the molecule responsible for the desired pharmacological action.
Though many albumin binding molecules are thus known, the vast majority of known albumin binders are not “portable”, in the sense that they cannot easily be coupled to other bioactive moieties without loss of albumin binding or other relevant pharmaceutical properties. Bromophenol blue and ibuprofen, two very well known molecules in biochemistry and in pharmaceutical sciences, may serve as examples to illustrate the case. Bromophenol blue is an avid albumin binder that forms complexes with albumin which are stable when analyzed by native polyacrylamide gel electrophoresis. However, the relatively complex chemical structure of bromophenol blue lacks suitable functional groups for the easy coupling of this molecule to other molecules of pharmaceutical interest (e.g., small organic drugs or protein drugs). Ibuprofen, one of the most widely used anti-inflammatory agents, binds to albumin with a dissociation constant in the micromolar range. However, as soon as one attempts to modify the carboxylic acid group of the molecule (e.g., by amide bond formation), in order to couple ibuprofen to other moieties of biopharmaceutical interest, ibuprofen loses its albumin-binding affinity.
The long circulation time of albumin has, nonetheless, been exploited for the direct conjugation of drugs to albumin. For example, a number of therapeutic polypeptides have been fused to albumin (EPO, interferons, interleukins, therapeutic peptides) at the genetic levels, and some of the corresponding fusion proteins are being investigated in the clinic [1].
An example is the direct coupling of chemotherapeutic agents to a unique cysteine residue (Cys34) on the surface of human serum albumin. Kratz and colleagues have used either the primary amino group or the carbonyl group of the anti-cancer chemotherapeutic drug doxorubicin for a site-specific coupling of this drug to the Cys34 residue of human albumin. The chemical moieties used as linkers between doxorubicin and albumin were chosen for their ability to be hydrolyzed, thus liberating the pharmacologically active doxorubicin moiety [2]. As a representative example, the maximum tolerated dose of DOXO-EMCH is 2-10 times higher than that of unmodified doxorubicin, and DOXO-EMCH can be injected at 2-10 times the molar equivalent of doxorubicin in tumor-bearing mice, with lower toxicity compared to unconjugated doxorubicin and more potent therapeutic effects. Similar results have been reported with the preformulated doxorubicin-HSA conjugate [3]. This protein derivative is currently being investigated in the clinic [4]. In a similar fashion, the same group has chemically coupled methotrexate to albumin, using a peptide linker which can be preferentially cleaved by matrix metalloproteinases at sites of arthritis in vivo. The corresponding protein-drug conjugate can be considered a prodrug and is currently in pharmaceutical development.
However, a severe general disadvantage of protein-drug conjugates is that they may be immunogenic in humans [5]. Direct conjugation can therefore not be considered a general solution to the problem of finding a means of associating drugs with albumin and thus improving their pharmaceutical properties. A generally applicable solution would be provided by portable albumin binding molecules, i.e. molecules that, on the one hand, have a selective affinity for albumin, and on the other hand may be attached to any drug of interest.
One of the physiological roles of albumin is to act as a carrier of fatty acids. This property has been exploited in order to associate a drug with albumin. For example, a well-known insulin derivative in commerce features a covalent modification of the insulin moiety by amide formation with tetradecanoic acid LEVEMIR®. This long fatty acid chain confers certain beneficial properties to insulin, including prolonged blood clearance, as would be expected considering the fatty acid-binding properties of albumin. However, the disadvantage of this approach is that it is not easily applicable to other molecules. In particular, it is not applicable to less soluble proteins or small drugs, due to the unacceptably low water solubility of the resulting derivative.
Recently, Genentech scientists have reported the discovery and use of disulfide-constrained albumin-binding peptides as a portable tag for altering the pharmacokinetic properties of proteins of biopharmaceutical relevance (e.g., antibody fragments), conferring them long blood circulation time in blood [6]. In a similar fashion, several biotech companies (e.g., Domantis, Affibody) have recently developed mutants of small globular proteins as “portable” albumin binders, in order to confer slow blood clearance properties to pharmaceutical proteins of interest, by genetic fusion [7-10]. However, peptides and proteins are not ideal as vehicles to confer albumin affinity upon drugs. Peptides and proteins are labile molecules that may be destroyed by digestion or denaturation, and are thus, in particular, difficult to administer orally. Such drugs must therefore in general be administered by injection, which is a cause of inconvenience compared to the oral route.
Apart from the field of drugs as such, there is also a need to control and improve the blood retention time and tissue distribution of diagnostic agents, such as diagnostic imaging and contrast agents. Fluorescein is a good example to illustrate such a need. Fluorescein is routinely used in opthalmology for fluoroangiographic imaging procedures, with over a million injections per year in the US and in Europe. Quite surprisingly, fluorescein is not an approved pharmaceutical agent. In most cases, a 3-5 ml fluorescein sodium solution in water (10%) has to be prepared by the physician prior to intravenous injection.
Fluorescein is cleared from the blood very rapidly, and therefore has to be administered at high doses, by bolus injection. However, at such high doses, fluorescein induces nausea and vomit in up to 20% of patients, depending on the patient group. These complications are more frequent in patients from black, Asian, Chino-Asian or mixed ethnic origins [11]. More severe reactions are rare, but include hives, laryngeal edema, bronchospasm, syncope, anaphylaxis, myocardial infarction and cardiac arrest [12].
Extravasation of fluorescein dye during the injection can be a serious complication of angiography. With a pH of the solution of 8 to 9.8, fluorescein infiltration can be quite painful. Sloughing of the skin, localized necrosis, subcutaneous granuloma and toxic neuritis have been reported following extravasation of fluorescein. Although life-threatening reactions during angiography are rare, the possibility that they may arise necessitates that angiographic facility should be properly equipped and prepared to manage serious reactions to the procedure.
Decreasing rate of injection may reduce the risk or severity of side reactions. However, this impairs imaging sensitivity.
There is therefore, e.g., a need for a derivative of fluorescein which is better and longer retained in the vascular space and is, in particular, less prone to either becoming metabolised or extravasation. Such a derivative would allow the administration of lower dosages, thus decreasing the likelihood and severity of side effects and complications, and would moreover lead to improved image quality.
Similar considerations apply to magnetic resonance imaging (MRI) contrast agents. Current extracellular MRI contrast agents are limited by relatively short half-lives and are also freely extravasated into background muscle, which decreases the contrast-to-noise ratio during steady-state imaging. For routine clinical use in MRI and magnetic resonance angiography applications, it is desirable that the contrast agent be selectively retained in the vascular space and have an extended blood half-life in order to provide a sufficient plasma concentration over a 1 hour imaging window. An extended blood half life would be necessary in order to facilitate imaging of multiple body regions and the acquisition of long, pulse-gated scans of coronary arteries [13].
A number of groups have aimed at improving the pharmacokinetic properties of contrast agents such as Gd-DTPA MAGNEVIST®, by coupling this moiety to portable albumin binders. However, the dissociation constants of such albumin binders have been limited to the order of 100 μM [14]. There thus remains a need for improving contrast agents such as Gd-DTPA, e.g., in order to extend their half-lives and improve their relaxation properties.
There is thus considerable interest in the identification and development of small organic molecules which may be used as portable albumin binding moieties for coupling to a wide range of molecules of pharmaceutical interest (e.g., small organic molecule drugs, protein drugs, or diagnostic agents, e.g., for various forms of medical imaging). However, the success of attempts to identify small organic molecules which may be used as portable albumin binders [15], has been limited so far. Particular areas of concern are the strength of binding to albumin, the chemical stability of albumin-binding conjugates in-vivo and the extent of portability (in particular, good pharmacokinetic properties of the conjugate should ideally be achievable with any drug or class of drugs, not only with certain favourable examples of drugs).
There is a need to minimize the size of portable albumin binders in order to allow the development of pharmaceutical agents which do not violate Lipinski's rule of five [16]. Other desired properties of an portable albumin binder include binding selectivity (i.e., discrimination between albumin and other molecules), easy synthetic accessibility, facile conjugation to (bio)pharmaceutical molecules of interest, good water solubility and absence of inherent toxicity. To date, it has not been possible to provide albumin binders that combine these advantageous properties.